International Journal of Molecular Sciences Article Isolation and Quantification of Uremic Toxin Precursor-Generating Gut Bacteria in Chronic Kidney Disease Patients Tessa Gryp 1,2,3,* , Geert R.B. Huys 3 , Marie Joossens 3 , Wim Van Biesen 1, 1, 2, Griet Glorieux y and Mario Vaneechoutte y 1 Department of Internal Medicine and Pediatrics, Nephrology Section, Ghent University Hospital, 9000 Ghent, Belgium; [email protected] (W.V.B.); [email protected] (G.G.) 2 Department of Diagnostic Sciences, Laboratory Bacteriology Research, Ghent University, 9000 Ghent, Belgium; [email protected] 3 Department of Microbiology, Immunology and Transplantation, Molecular Microbiology—Microbiome Research Lab, KU Leuven, 3000 Leuven, Belgium; [email protected] (G.R.B.H.); [email protected] (M.J.) * Correspondence: [email protected]; Tel.: +32-9-332-33-64 Equally contributing seniors. y Received: 26 February 2020; Accepted: 11 March 2020; Published: 14 March 2020 Abstract: In chronic kidney disease (CKD), impaired kidney function results in accumulation of uremic toxins, which exert deleterious biological effects and contribute to inflammation and cardiovascular morbidity and mortality. Protein-bound uremic toxins (PBUTs), such as p-cresyl sulfate, indoxyl sulfate and indole-3-acetic acid, originate from phenolic and indolic compounds, which are end products of gut bacterial metabolization of aromatic amino acids (AAA). This study investigates gut microbial composition at different CKD stages by isolating, identifying and quantifying PBUT precursor-generating bacteria. Fecal DNA extracts from 14 controls and 138 CKD patients were used to quantify total bacterial number and 11 bacterial taxa with qPCR. Moreover, isolated bacteria from CKD 1 and CKD 5 fecal samples were cultured in broth medium supplemented with AAA under aerobic and anaerobic conditions, and classified as PBUT precursor-generators based on their generation capacity of phenolic and indolic compounds, measured with U(H)PLC. In total, 148 different fecal bacterial species were isolated, of which 92 were PBUT precursor-generators. These bacterial species can be a potential target for reducing PBUT plasma levels in CKD. qPCR indicated lower abundance of short chain fatty acid-generating bacteria, Bifidobacterium spp. and Streptococcus spp., and higher Enterobacteriaceae and E. coli with impaired kidney function, confirming an altered gut microbial composition in CKD. Keywords: qPCR; bacterial culture; fecal bacteria; chronic kidney disease; protein-bound uremic toxins; bacterial metabolization; aromatic amino acids; p-cresol; indole; indole-3-acetic acid; phenol 1. Introduction In chronic kidney disease (CKD), impaired kidney function results in the accumulation of uremic toxins [1–3], which exert deleterious biological effects and contribute to inflammation and cardiovascular morbidity and mortality [4–8]. Well-known protein-bound uremic toxins (PBUTs), p-cresyl sulfate (pCS), p-cresyl glucuronide (pCG), indoxyl sulfate (IxS) and indole-3-acetic acid (IAA) originate in the colon, where aromatic amino acids (AAA) are metabolized by gut bacteria into phenolic (p-cresol and phenol) and indolic compounds (indole and IAA) [9–12]. p-Cresol and indole are sulfated in the colon mucosa and by the liver into pCS and IS, and a small part is glucuronidated into pCG [9,13,14], Int. J. Mol. Sci. 2020, 21, 1986; doi:10.3390/ijms21061986 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 1986 2 of 19 while IAA enters the circulation without further modifications [15–17]. These PBUTs reversibly bind to plasma albumin [18]; however, only the free fraction can be removed by dialysis [19,20]. During recent years, there is increasing evidence that the gut bacterial composition is altered in patients with CKD [21–30]. Generally, in CKD, increased growth of both aerobes and anaerobes takes place in the small intestine [24,25], while in the colon the numbers of aerobic bacteria (e.g., Enterobacteriaceae) are increased [23,30] and anaerobic bacteria (e.g., bifidobacteria) are decreased [23,29,30]. The increased aerobic bacterial growth is a consequence of an elevated pH resulting from the high ammonia levels in the gut in CKD [31]. Ammonia is a bacteria-derived hydrolysis end product of urea, which accumulates in the circulation and enters the gut lumen via the entero-hepatic cycle [31] and by diffusion through the disrupted intestinal epithelial barrier as is typically seen in patients with CKD [32,33]. This leads to a uremic milieu in the gut lumen, altering the gut microbial composition in favor of urease expressing bacteria in end stage kidney disease (ESKD) patients [27]. However, in a recent study by our group [34], the generation of fecal PBUT precursor metabolites by fecal bacteria did not alter with CKD progression nor compared to controls. This suggests that the accumulation of PBUTs in plasma is mainly due to the impaired kidney function itself. Nevertheless, minimizing the generation by gut bacteria of phenolic and indolic compounds in the hope to subsequently reduce plasma PBUT levels remains an important goal due to the absence of other adequate therapies to reduce the plasma levels of PBUTs in patients with CKD. It is known that certain bacterial isolates are capable to generate PBUT precursors in an in vitro setting [9,35–40], but the generation capacity of fecal bacteria isolated from patients with CKD has not yet been investigated. Numerous studies in patients with CKD already addressed the idea of applying pro-, pre- and synbiotics [30,41–46] to increase saccharolytic and decrease proteolytic bacteria. Thus far, only one specific PBUT has been targeted [47]. In addition, it has been shown that dietary intake is the most important determinant of the fecal metabolome in CKD, next to the loss of kidney function [13], suggesting that diet could also play an important role in altering the gut bacterial composition in a favorable way. Intake of low-protein diets lowers the serum levels of IxS and the generation of various phenolic and indolic metabolites in healthy humans and mice [48]. In this study, bacterial isolates obtained from fecal samples from patients with CKD stage 1 and stage 5 were taxonomically characterized and metabolically classified as PBUT precursor-generating bacteria based on their capacity to assimilate AAA into phenolic (p-cresol and phenol) and/or indolic (indole and IAA) compounds. In addition, the abundance of specific fecal PBUT precursor-generating bacteria in the different stages of CKD and in controls was investigated. 2. Results 2.1. Culture Media to Isolate Protein-Bound Uremic Toxin Precursor-Generating Bacteria from Fecal Samples To isolate PBUT precursor-generating bacteria, an appropriate culture medium had to be selected. Therefore, seven different media were compared based on the cultivation of a bacterial test panel consisting of 12 known uremic toxin precursor-generating bacterial species (Figure S1), and the cultivation of fecal bacteria from whole fecal samples (Table S1). Besides the commercially available Schaedler medium (SCH, Becton Dickinson, Erembodegem, Belgium), the yeast casitone fatty acid glucose (YCFAG) medium [37,49] was used as the basis for another six media, of which specific components were omitted separately, to check their importance for the cultivation of UT precursor-generating bacteria. Moreover, the effect of the supplementation of AAA and of a fecal suspension from healthy volunteers (FS) was investigated. All bacterial species from the test panel were culturable with the 24 YCFAG media combinations, except for Bifidobacterium sp. and Lactobacillus sp., which only grew when AAA and FS were added to the complete YCFAG (YCFAG D0), YCFAG without vitamins (YCFAG DV) or YCFAG without short chain fatty acids (YCFAG DS). For the SCH medium, addition of AAA was detrimental for the growth of most tested bacterial species, except B. longum, E. coli, L. paracasei, and S. epidermidis. SCH medium, Int. J. Mol. Sci. 2020, 21, 1986 3 of 19 with and without the supplementation of FS, resulted in the growth of all species of the test panel (Figure S1). Based on these results, the five medium formulations that supported growth of all bacterial species (i.e., YCFAG D0 +AAA +FS, YCFAG DV +AAA +FS, YCFAG DS +AAA +FS, SCH and SCH + FS) were used further to culture bacteria from fecal samples. Table S1 provides an overview of all microorganisms that could be cultured on these five media. Most anaerobic and aerobic bacteria were isolated from YCFAG D0 +AAA +FS and from SCH, and these media were further used to isolate PBUT precursor-generating bacteria from fecal samples of patients with CKD. 2.2. Isolation and Identification of Protein-Bound Uremic Toxin-Generating Bacteria from Fecal CKD Samples Figure S2 illustrates an overview of the isolation of PBUT precursor-generating bacteria. In total, 148 different bacterial species were isolated from CKD 1 (n = 6) and CKD 5 (n = 6) fecal samples to cover fecal bacteria representative for both early and late stage of CKD. Of these, 99 species could be identified by means of MALDI-TOF (Bruker, Mannheim, Germany) and 92 were considered as PBUT precursor-generating bacteria when, for at least one of the precursor metabolites (i.e., p-cresol, phenol, indole or IAA), the levels were above the limit of detection (LOD) as assessed by U(H)PLC. Table1 lists all isolated bacterial species with their generation capacity of PBUT precursors under aerobic and anaerobic conditions. Generally